NEW SEQUENCING

TECHNOLOGY

S@NDEEP YADAV
Content SOLEXA
• INTRODUCTION
• NEW SEQUENCING TECHNOLOGY- A
COMPARISION
• PYROSEQUENCING
• Roche/454 Technology
• ILLUMINA SOLEXA TECHNOLOGY
• REFRENCE
 INTRODUCTION 454 GS FLX
• Developing new methods for fast
polynucleotide sequencing
• Quick identification of pathogens to save
many lives
• Sequencing has important medical
applications
• Sequencing methods proposed by
Fredrick Sanger, Maxim & Gilbert
• Dideoxy method was accepted because of
easiness
• Human genome project cost 3 billion US
dollars and took 13 years to complete.
New Sequencing Technology

Roche 454 GS FLX Pyrosequencing

Illumina SOLEXA Sequencing by

synthesis
Applied Biosystems
SOLID Sequencing by
ligation
VisiGen
Biotechnologies Single-molecule
sequencing
Helicos BioSciences Nanopore sequencing
 SEQUENCING TECHNOLOGY- A
COMPARISION

Roche/454 ABI Solid Illumina/Solexa

Sequencing Pyrosequencing Oligo ligation- Labeled base

method chemistry cleavage addition

Base per run 100 million 1000-1500 million 1000-3000 million

Read length 100-200 bases 35 bases 35-50 bases

Length of run 7.5 hrs. 2-4 days 2-3 days

Cost of just $ 9,000 per run $ 5,000 per run $ 3,500 per run
sequencing
 PYROSEQUENCING
• Sequencing primer is hybridized to PCR
amplified, ssDNA template
• Incubated with DNA pol, ATP sulfurylase,
luciferase and apyrase, (substrate) adenosine 5´
phosphosulfate (APS) & luciferin.
• First four dNTP is added to the reaction
• DNA pol catalyzes the incorporation of
complementary base in the template strand
• Each incorporation event will release
pyrophosphate (PPi) in a quantity equimolar to
the amount of incorporated nucleotide
• APS + PPi ATP + Sulphate

•Luciferin + ATP oxyluciferin + visible light

•Light produced is detected by a charge coupled device

(CCD) camera and seen as a peak in a pyrogram™. Each
light signal is proportional to the number of nucleotides
incorporated.
PYROSEQUENCING

• Apyrase continuously degrades

unincorporated dNTPs. Now another set
of dNTPs added
• Note that deoxyadenosine alfa-thio
triphosphate (dATPS) substitutes the
natural deoxyadenosine triphosphate
(dATP).
• As the process continues, the
complementary DNA strand is built up
and the nucleotide sequence is
determined from the signal peak in the
Pyrogram® trace.
 454 Sequencing Instrument Ease of Use 3. Load Reagents
2. Load PicoTiter in a single rack
plate into instrument

4. Sequence Entire
Genome at once,
in real-time

1. Genome is loaded
into a PicoTiter™ plate
454 LifeSciences Sequencer
 454 LifeSciences Sequencer

GS FLX sequencer sequence the genomic DNA, PCR products, BACs and cDNA.
•Longer sequences are first sheared into random library of 300-800 bp long fragments.
•Adaptors added to both ends of the fragments. If sample is double stranded one strand is
removed and the remaining single strands are used in the following steps
•Aided by the adaptors individual fragments are captured on their own unique beads. A
bead and the bound fragment together with a water-in-oil emulsion form a microreactor so
that each fragment can be amplified without contamination via the so called emulsion PCR
(emPCR). The entire fragment collection is amplified in parallel via emPCR.
•Now the emulsion shell is broken and the clonally amplified beads are ready for
loading onto the fibre-optic PicoTiterDevice for sequencing.
 454 LifeSciences Sequencer

•The PicoTiter
Plate is loaded
with one
fragment
carrying bead
per well and
smaller beads
with the
enzymes
necessary for
sequencing.

•Sequencing is accomplished by synthesizing the complementary strands of the bead attached

templates. In a number of cycles the four bases (ATGC) are sequentially washed over the
PicoTiterPlate. The incorporation of a new base is associated with the release of inorganic
pyrophosphate starting a chemical cascade. This results in the generation of a light signal
which is captured by a CCD camera.
454 LifeSciences Sequencer

• Real Time Sequencing by Synthesis

• Chemiluminescence detection in picotiler plates
• Amplification: emulsion PCR
• Pyrosequencing
• up to 400,000 reads / run
• on average 250 bases / read
• up to 100 Mb / run
• 99% accuracy at the 400th base
• G-C rich content is not as much of a problem
• capable of detecting mutations in an amplicon
pool at a high sensitivity level, which may have
implications in clinical research, especially cancer
and HIV
ILLUMINA SOLEXA Flow Cell
ILLUMINA SOLEXA SEQUENCER
ILLUMINA SOLEXA SEQUENCER Contd.
Illumina / Solexa: Genetic Analyzer
PRO

• Real Time Sequencing by Synthesis

• Clonal Single Molecule Array
• Amplification: bridging PCR
• 60 mio reads / run
• up to 50 bases / read
• 2 Gb / run
• 8 channels, app. 5 mio reads / channel
• Fluorescent labels
• Reversible 3‘OH blocking
 APPLICATION OF GEMONE ANALYSER

• de novo sequencing
• cDNA libraries, ESTs
• amplicon sequencing, long range PCR fragments
• human samples
• BAC pools
• fosmid pools
• metagenomes, biofilm
• transcriptome
REFRENCE

• www.illumina.com
• www.en.wikipedia.org/wiki/DNA_sequencing
• www.ncbi.nlm.nih.gov/
• www.454.com
• www.genengnews.com/news
THANK YOU